Construction Of C5-Ketoreductase Conjugal Transfer Disruption Strain Of Streptomyces Bingchenggensis

The commercial product milbemycins A3and A4are16-membered macrolide antibiotics with outstanding activity against various kinds of mites.5-oxime derivatives of milbemycins A3and A4were found to be highly effective as anthelmintics.The C5position due to its keto-chemical properties of activity, it’s easy synthesis of5-oxime milbemycins derivatives, the chemical synthesis of5-oxime milbemycins the best precursor derivatives, therefore, it is important to elucidate the biosynthetic step from5-oxomilbemycins A3and A4to milbemycins A3and A4respectively.Streptomyces bingchenggensis that produced milbemycin and bingchengmycin is a new strain of Streptomyces discovered by our laboratory, the metabolites of Streptomyces bingchenggensis have better insecticidal activity and high control capacity.Conjugal transfer, a powerful trans-gene tool, has been successfully applied in various Streptomyces stains for genetic manipulation. This method protects foreign DNA from nuclease-induced degradation thereby avoiding restriction of host cells and largely enhancing the gene transfer efficiency.binF is a gene encoded transferase that catalyzed C5position of macrolide antibiotics into ketonic group, presenting conserved sequence in Streptomyces bingchenggensis. In this study, we used the methods of molecular biology to made binF gene inactivation, in order to get a C5-oxomilbemycins producing strains.We made the Streptomyces bingchenggensis226541as the experimental material, conjugative transfer system of Streptomyces bingchenggensis was used and the disruption of binF in Streptomyces bingchenggensis strain was attempted.To further study function of the gene in Streptomyces bingchenggensis, analyzing the bio-information of binF genomic sequence. Firstly, primer binF-Ll, binF-L2and binF-R1, binF-R2were designed to successfully amplified5’flank and3’flank fragments of binF from Streptomyces bingchenggensis, pBC810was constructed by connected the two fragments and tsr with pMD19-T Vector, then used the gene disruption plasmid pKC1139to build the homologous recombination vector pBC3784and put pBC3129into ET12567(pUZ8002), and obtain conjugative transfer donor strains ET12567(pUZ8002/pBC3129) through conjugative transfer into Streptomyces bingchenggensis. Under the antibiotic selection pressure and PCR testing, transformants isolated phenotype for ThioRAms of the mutant with binF gene disruption. Transformants were verified to right. The conjugative transfer system of Streptomyces bingchenggensis226541and E.coli strain ET12567(pUZ8002/pBC3129) was developed. Streptomyces bingchenggensis spores were28℃incubated30min, the OD600of E.coli strain ET12567(pUZ8002) was about0.2, mixed and painted in MS medium that contained30mM MgCl2, the time of antibiotic coverage for16h.